Moderate bleeding for organ resection, some types of trauma, bone fractures, orthopedic procedures, vascular anastomosis, solid organ wounds, and deep dermal wounds often require an adjunct to control the bleeding before or after sutures or stitches are applied.
Current solutions and limitations. Various technologies have been developed for the formulation of hemostats. Some of them contain biological materials such as collagen or fibrinogen and thrombin. As a result of their hemostatic and adhesive properties, fibrin sealants, or thrombin-based products have been extensively used in most surgical specialties for over two decades to reduce blood loss and post-operative bleeding due to their ability to adhere to human tissue as it polymerizes (1, 2, 3). These compounds are used to seal wounds that have been sutured or stapled; they can also be used with pressure over an injured area to control bleeding. Fibrin sealants are biological adhesives that mimic the final step of the coagulation cascade. (4) The main components of the sealant are plasma proteins fibrinogen, and factor XIII on the one hand and thrombin, and calcium chloride on the other. The components are often extracted from human plasma or produced by recombinant techniques. Mixing fibrinogen and thrombin creates a polymer barrier (fibrin) that simulates the last stages of the natural coagulation cascade to form a structured fibrin clot similar to a physiological clot. Products containing thrombin stimulates the coagulation cascade forming a natural fibrin clot.
There are several commercial products available (Floseal, Gelfoam, Evicel, Bioglue) (3, 5). However, these products have limitations such as inflammatory effects, immunological responses, storage conditions, preparation, and need of compression. Most hemostatic agents for intracavitary bleeding are designed to be used in light to moderate bleeding in the operating room and require hard compression. One of the major limitations encountered in the development and/or use of fibrin compositions with brief or no compression is their inability to form a sufficiently strong bond to tissues, to produce a stable clot within minutes of application, and the need to incorporate thrombin and anti-fibrinolitic agents in the formulation. However, there are many situations where the use of strong compression, sutures and/or staples is undesirable, inappropriate or impossible, e.g. in bone, brain, interventional radiology, retroperitoneal surgery. Also the inclusion of thrombin prevents the recover of lost blood.
The present alternative approach: In order to form a physical barrier that resists the flow of blood, the adhesive matrix of fibrin-based hemostat must form in a matter of seconds a strong fibrin interface, bond with tissues in the midst of flowing blood and remain at the lacerated site to form a clot. The ability of ClotGel, produced as a dense colloidal solution similar to a gel, to adhere to human tissue is related to the use of desAB fibrin monomer described in U.S. Pat. No. 8,367,802 by G Falus et al., and its physical properties such as density, composition and method of production of fibrin. Unlike other fibrin sealants that use thrombin to cleave fibrinogen in situ to form a desAB fibrin II (fibrin II) polymer, ClotGel polymerizes desAB fibrin monomer by neutralization in contact with the blood, thus bypassing the cleavage process. The essential aspect of the technology is the ability of crosslinked desAB fibrin (fibrin II) polymer and desAB firbrin (fibrin II) monomer to be mixed with the blood -to form a physical barrier that turns into a functional fibrin clot. (6) In our approach these results are obtained through the in-situ generation of a three-dimensionall polymeric cross-linked fibrin network that is bonded to the tissue as a strong fibrin clot stabilized by Factor XIII present in the blood.
The crosslinked fibrin incorporated into the monomer solution allows to produce a viscous substance that resist the flow of blood, and facilitates transportation and readiness.
Composition. The present technology incorporates crooslinked fibrin polymer produced from neutralized desAB fibrin (fibrin II) monomer, which is then lyophilized, homogenized in neutral buffer, transferred to acid solution, and mixed with fibrin solution kept at pH 3.4. The homogenized polymer-monomer mixture is ready to polymerize at change of pH when in contact with the blood. The desAB fibrin (fibrin II) monomer is produced by the dialysis method (U.S. Pat. No. 8,367,802 by G Falus et al.). The lyophilized crosslinked desAB fibrin (fibrin II) polymer is produced by neutralizing the fibrin monomer with a neutral buffer solution containing calcium chloride, crosslinking with calcium-dependent Factor XIII and calcium-independent transglutaminase enzymes and subsequent Lyophilization.
Mixing blood with the gel-like composition creates a mesh of fibrin fibers that form the fibrin clot at sites of injury (7). Under coagulant conditions, calcium-independent transglutaminase enzyme and activated Factor XIII contribute to this process by stabilizing the fibrin clot through covalent bonds.
Key Attributes. Polymerization/Adhesion. The fibrin gel that seals the wound is formed as a result the absorption of blood, and pH neutralization of the monomer in contact with wound that rapidly turns into polymer of long fibers, trapping the-lyophilized/homogenized fibrin polymer material, which acts as a biocompatible support. The transglutaminase enzyme in the lyophilized component and Factor XIII in the blood leads to further formation of covalent bonds in the clot. The clot is mechanically stable, well integrated into the wound and more resistant to lysis by plasmin compared with uncrosslinked clots [8] or other fibrin sealants. The inclusion of calcium-independent transglutaminase enzyme facilitates the transglutaminase-mediated oligomerization of the αC-domains of fibrin promoting integrin clustering and thereby increasing cell adhesion and spreading, which stimulates fibrin to bind αvβ3-, αv-β5-and α5-β1-integrins on endothelial cells (9). The oligomerization also promotes integrin-dependent cell signaling via focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK), which results in an increased cell adhesion and cell migration [10].
The adhesion characteristics to vital human tissue and the kinetics of polymerization of the proposed agent have been tested in vitro and in vivo. The data obtained provide ample evidence of the ability of ClotGel to stop bleeding and achieve hemostasis with and without compression in four surgical protocols including vascular anastomosis, knee replacement, liver and kidney laceration/resection and spleen lacerations in the swine models. The polymerization process begins within 6 seconds of application.
Fibrin Monomer Polymerization:
U.S. Pat. No. 8,367,802 by Falus et al. describes a method of preparing a fibrin monomer. The ClotGel sealant composition uses a lyophilized crosslinked fibrin polymer obtained by neutralizing of fibrin monomer, which is mixed with fibrin monomer. The composition of parts and method of production of the fibrin monomer described in this patent application as well as the process of neutralization and crosslinking of the polymer are critical to the performance of the proposed technology, which depends on the characteristics of fibrin itself (thickness of the fibers, the number of branch points, the porosity, and the permeability. The clot produced by ClotGel creates opaque matrices of thick fibers, and, therefore, formation proceeds at a faster rate than in transparent matrices. The concentration of thrombin to produce a fibrin monomer also has an important impact on the polymerization process.
ClotGel Presentations:
The fibrin suspension can be presented in several dosages, from 5 cc to 20 cc in order to adapt to the type of application (FIG. 1).